A series of conformational rearrangements of the spliceosome affects its assembly, catalysis, and disassembly steps. We have recently formulated a model of spliceosome function, according to which structural conformations of the complex that define the two catalytic steps exist in equilibrium with each other (Query and Konarska, 2004). Our model suggests remarkable similarities between the mechanisms of function of the ribosome and the spliceosome. In both cases, equilibrium between conformational states can be modulated by several factors; the transition is facilitated by the action of EF-Tu GTPase or Prp16 DEAH- box ATPase, but specific contacts between the RNA substrate (i.e. tRNA or pre-mRNA) and the enzyme (i.e. RNP complex) also contribute to this transition through an induced fit mechanism. We propose several lines of experiments, all based on a similar premise - that individual steps in the splicing pathway are in equilibrium with the adjacent steps. In Aim 1, we will test the hypothesis that 5'SS-U6 and BS-U2 RNA-RNA interactions become disrupted at the stage of transition from the 1st to 2nd step of splicing. We will prepare a set of substrates capable of forming either destabilized or hyperstabilized pairing interactions with U6 and U2 snRNAs, and test their effects on the efficiency of the 1st and 2nd steps. We will also test if the function of prp16 mutants defective in the transition between the catalytic steps is further inhibited by such hyperstabilized substrates. The 3' exon is likely to contain positions that improve the 2nd step efficiency, perhaps representing the exon binding site for the 2nd step. In Aim 2, we will search for such 3' exon positions and characterize their interactions at the catalytic center. We will also search for positions at the 3' end of the intron that influence 2nd step efficiency. We have previously identified a number of prp8 alleles that improve the 2nd step of splicing, and inhibit the 1st step. In Aim 3, we will identify and characterize members of the opposite lass of prpS alleles that improve the 1st step, but inhibit the 2nd step. We will also test if the 1st and 2nd step suppressors cancel each other's effect. In Aim 4, we will analyze the involvement of Prp2 DEAH-box ATPase in the transition preceding the 1st step of splicing. Selection of cryptic 5'SS will be used to monitor activity of prp2 alleles defective in transition into the 1st step conformation of the spliceosome.